Abstract
Mitochondrial localization of the radioprotective MnSOD transgene in hematopoietic cells dismutates irradiation induced superoxide to hydrogen peroxide which is converted to water and oxygen by catalase or glutathione peroxide. Increased concentration of hydrogen peroxide can be toxic. We hypothesized, that increased mitochondrial localized catalase to remove hydrogen peroxide would further increase radioresistance. The human catalase transgene was cloned into a pSVZeo plasmid. To localize the transgene product catalase to the mitochondria, the mitochondrial localization sequence of MnSOD was cloned and attached to the catalase transgene (mt-catalase), then cloned into a pSVZeo plasmid. The plasmids were electroporated into murine hematopoietic cell line 32Dcl3 and 32Dcl3 MnSOD transgene overexpressing clonal cell line 2C6 and subclones of each expressing the non-targeted catalase or mt-catalase selected by growing the cells in zeomycin. The clonal cell lines were shown to express either catalase or mt-catalase by RT-PCR using specific primers. Catalase biochemical activity was determined and 32D-cat and 32D-mt-cat cells had increased catalase activity (595.7 ± 15.3 or 603.3 ± 3.0 μM, respectively) compared to 539.7 ± 3.7 μM (p = 0.0288 or 0.0002, respectively) for 32Dcl3. Compared to the 32Dcl3 cells, 2C6 cells had decreased catalase activity of 205.0 ± 10.0 compared to 539.7 ± 3.7 (p < 0.0001). Catalase activity was increased in 2C6-cat and 2C6-mt-cat (333.3 ± 12.7 and 467.0 ± 1.0, respectively) compared to 205.0 ± 1.0 for 2C6 (p <0.001). Western analysis confirmed the differences in catalase activity. Cells from 32Dcl3, 32D-cat, 32D-mt-catalase, and subclones of 2C6 cells were irradiated to doses ranging from 0 to 8 Gy, plated in methylcellulose, incubated at 37° C for seven days, and colonies of greater that 50 cells counted. The data was analyzed by linear quadratic and single-hit, multi-target models. The 32D-mt-cat cells were more radioresistant than 32D-cat cells by an increased shoulder on the survival curve (n = 10.3 ± 0.5 or 5.9 ± 0.2, respectively, p = 0.0025). Both 32D-mt-cat and 32D-cat were more resistant compared to 32Dcl3 cells (n = 2.9 ± 1.1, p = 0.0196 or 0.0479). Cells from the 2C6 transfected with mt-catalase, but not catalase, showed increased radioresistance increasing the Do from 0.979 ± 0.1Gy for 2C6 to 1.171 ± 0.1 Gy for 2C6-mCat cells. To determine if increased catalase activity altered antioxidant status, levels of glutathione (GSH) and glutathione peroxidase (GPX) were measured. There was no significant change in GSH between cell lines. However, there were increased levels of GPX in 32D-cat and 32D-mt-cat of 260.4 ± 24.6 or 257.1 ± 17.1 μM, respectively, compared to 105.5 ± 1.6 μM (p = 0.0005 or 0.0134, respectively). Cells from 2C6, 2C6-cat or 2C6-mt-cat had decreased GPX activity to 46.7 ± 1.3, 39.1 ± 0.9 or 44.1 ± 1.5μM, respectively, compared to 105.5 ± 1.6μM for 32Dcl3 (p<0.0001). Thus, overexpression of both MnSOD and mt-catalase transgene provides superior radioprotection compared to one alone.
Microbial resistance in relation to catalase activity to oxidative stress induced by photolysis of hydrogen peroxide